Project Summary Chronic arsenic exposure is a worldwide public health concern correlated with an increased risk of developing certain types of cancer, as well as metabolic diseases including type II diabetes. Arsenic-linked type II diabetes has recently been shown among populations in the United States, Mexico, Canada, Taiwan, and Bangladesh. We have previously reported that low, environmentally relevant doses of arsenic block autophagy at a later stage. Autophagy is a key cellular degradation pathway and autophagic dysfunction is thought to be an integral part of pathogenic changes that occur in adipocytes, ?-islet cells, hepatocytes, skeletal muscle, and kidney mesangial cells during diabetes progression. We have also shown that dysregulation of the autophagy pathway results in prolonged activation of the key antioxidant transcription factor nuclear factor erytheroid-derived-2-like 2 (Nrf2). Nrf2, which is normally bound and degraded in the cytosol by Kelch-like ECH-associated protein 1 (Keap1), is upregulated at the protein level following oxidative modification of Keap1 (Keap1-C151 dependent, canonical) or by sequestration of Keap1 into autophagosomes during autophagy dysfunction (p62-dependent, non-canonical). While controlled Nrf2 activation through the canonical mechanism is protective, prolonged non-canonical activation of Nrf2 causes cellular dysfunction and tissue damage, indicative of a ?dark side? to Nrf2. Therefore, we hypothesize that the prolonged activation of Nrf2, resulting from arsenic-mediated blockage of autophagy flux, is essential for arsenic in promoting type II diabetes. This hypothesis is supported by the following evidence: 1) chronic arsenic exposure decreases glucose uptake and insulin signaling in 3T3-L1 adipocytes, 2) Keap11 KD/Lepob/ob mice, a genetic mouse model for diabetes with persistent Nrf2 activation, display impaired adipogenesis, as well as decreased insulin sensitivity and glucose tolerance compared to Lepob/ob controls, and 3) ?-cell specific knockdown of Atg7, a key autophagy initiation protein, results in increased levels of p62 and poly-ubiquitinated proteins, accompanied by ?- cell loss and decreased insulin production. We have generated a large amount of data indicating that arsenic blocks autophagy at the autophagosome-lysosome fusion step. Three core SNARE proteins Stx17, SNAP29, and VAMP8 mediate fusion, with SNAP29 mediating the interaction between Stx17 on the outer autophagosomal membrane and VAMP8 on the lysosomal membrane. We believe that genetic ablation of any of these three fusion proteins will hinder autophagosome-lysosome fusion and result in prolonged Nrf2 activation, which will mimic the effect of arsenic in promoting type II diabetes. Therefore, we propose to: Aim 1: Determine the molecular mechanism by which arsenic blocks autophagosome- lysosome fusion, leading to prolonged Nrf2 activation. Aim 2: Determine if prolonged Nrf2 activation resulting from autophagic dysfunction induces metabolic reprogramming in muscle, kidney, pancreas, liver and fat cells. Aim 3: Determine if autophagy dysfunction and prolonged Nrf2 activation phenocopy arsenic in exacerbating insulin resistance, obesity, and diabetic nephropathy using type II diabetes models in SNAP29f/f, Nrf2-/-, and SNAP29f/f/Nrf2-/- mice. Impact: A detailed and thorough understanding of autophagy dysfunction and the prolonged activation of Nrf2 in arsenic-induced metabolic disease will prove extremely valuable in the generation of preventive and therapeutic strategies, as well as in the identification of biomarkers, for the populations at risk.